This invention relates to novel polyunsaturated fatty acid derivatives and their use as therapeutic agents.
Amides of fatty acids (saturated and unsaturated) having the following formula:
Rxe2x80x94COxe2x80x94NH2
wherein the R is alkyl residue of fatty acid, represent a new group of lipid bioregulators originated from long-chain fatty acids via amidation by corresponding amines. Erucamide (13-docosenamide) was found to be the major bovine mesentery angiogenic lipid. The mechanism of angiogenic activity is unknown and this lipid does not promote proliferation of endothelial cells or induce inflammatory effects (Wakamatsu K. et al., Biochem. Biophys. Res. Commun., V.168. p. 423-429, 1990). A molecule isolated from the cerebrospinal fluid of sleep-deprived cats has been chemically characterized and identified as cis-9,10-octadecenoamide. Other fatty acid primary amides in addition to cis-9,10-octadecenoamide were identified as natural constituents of the cerebrospinal fluid of cat, rat and human, indicating that these compounds compose a distinct family of brain lipids. Synthetic cis-9,10-octadecenoamide induced physiological sleep when injected into rats. Together, these results suggest that fatty acid primary amides may represent a previously unrecognised class of biological signal molecules (Cravatt B. F. et al., Science., V. 268., P.1506-1509, 1995).
A very thoroughly investigated group of compounds are ethanol amides of fatty acids, having the following formula:
Rxe2x80x94COxe2x80x94NHxe2x80x94CH2CH2xe2x80x94OH
where R is an alkyl residue of a fatty acid.
Ethanol amides of fatty acids bound with cannabinoid receptors in the central nervous system or in peripheral tissues are considered as endogenous ligands for these receptors. They exhibit pharmacological patterns like cannabimimetics (Bezuglov V. V. et al. Biochemistry (Moscow). V.63, N 1. P. 27-37,1998).
Amides of retinoic acid (cis-trans isomers) with some amino acids having the following formula:
Rxe2x80x94COxe2x80x94NHxe2x80x94Yxe2x80x94COOH
wherein R is an alkyl residue of retinoic acid and Y is a residue of amino acid have been synthesised via all-trans-retinoyl chloride and an ester of the amino acid (Shealy Y. F. et al., J.Med.Chem. V.31.,P.190-196, 1988). The retinoyl derivatives of leucine, phenylalanine, alanine, tyrosine and glutamic acid were prepared. The 13-cis-retinoyl derivatives of leucine, phenylalanine, alanine, and glycine were prepared similarly from 13-cis-retinoic acid. In assays of the retynoylamino acids for reversal of squamous metaplasia in hamster trachea organ cultures, these compounds were less active than retinoic acid, but the leucine, alanine and phenylalanine derivatives were similar in activity to several retinamides that suppress bladder carcinogenesis in vivo. Two of the retinoylamino acids, as well as two simple retinamides were shown to be moderately cytotoxic to murine leukemia and human epidermoid carcinoma cells in culture.
One problem addressed by the present invention is that of making available novel compounds for use in the manufacture of pharmaceuticals, e.g. for the treatment of cancer and immune deficiencies, said compounds exhibiting i.a. higher activity than presently known compounds.
The present invention makes available novel amides according to the attached claims, in particular amides of the all-trans-retinoic acid or 13-cis-retinoic acid and arachidonic acid and docosahexaenoic acid and eicosapentaenoic acid or linoleic acid with 2-aminoethanol, alpha-L-serine, alpha-L-threonine, alpha-L-tyrosine containing phosphate groups. Further, the present invention discloses the use of these compounds, in particular their pharmaceutical application as specified in the attached claims.
The structure of these compounds is covered by the following general formula:
xe2x80x83Rxe2x80x94CONHxe2x80x94Xxe2x80x94OPO(OH)2
wherein
R is 
or CH3(CH2)4(CHxe2x95x90CHxe2x80x94CH2)4CH2CH2xe2x80x94
or CH3(CH2CHxe2x95x90CH)6CH2CH2xe2x80x94
or CH3(CH2CH2CHxe2x95x90CH)5(CH2)3xe2x80x94
or CH3(CH2CHxe2x95x90CH)3(CH2)7xe2x80x94
and X is xe2x80x94CH2xe2x80x94CH2xe2x80x94
or xe2x80x94CH(CO2H)xe2x80x94CH2xe2x80x94
or xe2x80x94CH(CO2H)xe2x80x94CH(CH3)xe2x80x94
or xe2x80x94CH(CO2H)xe2x80x94CH2xe2x80x94C6H5xe2x80x94
Thus the amino group of 2-aminoethanol or the alpha-amino group of amino acid forms an amide bond with the carboxylic group of arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid or linoleic acid and all-trans-retinoic acid or 13-cis retinoic acid. At the same time the hydroxyl group of 2-aminoethanol and the amino acid is modified by a phosphate residue. The compounds can be applied as immunostimulating therapeutical agents for the treatment of immune-deficiencies.
The novel compounds according to the present invention are amides of all-trans-retinoic acid or 13-cis-retinoic acid and arachidonic acid and docosahexaenoic acid and eicosapentaenoic acid or linoleic acid with 2-aminoetanol, alpha-L-serine, alpha-L-threonine, alpha-L-tyrosine. At the same time hydroxyl groups of amino acids and 2-aminoethanol are modified by phosphate residues. The all-trans-retinoic acid or 13-cis retinoic acid and arachidonic acid and docosahexaenoic acid and eicosapentaenoic acid or linoleic acid have been derived by various procedures from naturally-occurring products. It is however possible, within the scope of the present invention, to produce these compound synthetically.
The main characteristic among the novel synthesised compounds is the phosphorylation of the hydroxyl groups of N-acyl derivatives of amino acids and 2-aminoethanol.
Retinoic acid derivatives according to the present invention include the following compounds:
1. N-(all-trans-retinoyl)-o-phospho-2-aminoethanol
1a. N-(13-cis-retinoyl)-o-phospho-2-aminoethanol
2. N-(all-trans-retinoyl)-o-phospho-L-serine
2a. N-(13-cis-retinoyl)-o-phospho-L-serine
3. N-(all-trans-retinoyl)-o-phospho-L-threonine
3a. N-(13-cis-retinoyl)-o-phospho-L-threonine
4. N-(all-trans-retinoyl)-o-phospho-L-tyrosine
4a. N-(13-cis-retinoyl)-o-phospho-L-tyrosine
Arachidonic acid derivatives according to the present invention include the following compounds:
5. N-arachidonoyl-o-phospho-2-aminoethanol
6. N-arachidonoyl-o-phospho-L-serine
7. N-arachidonoyl-o-phospho-L-threonine
8. N-arachidonoyl-o-phospho-L-tyrosine
Docosahexaenoic acid derivatives according to the present invention include the following compounds:
9. N-docosahexaenoyl-o-phospho-2-aminoethanol
10. N-docosahexaenoyl-o-phospho-L-serine
11. N-docosahexaenoyl-o-phospho-L-threonine
12. N-docosahexaenoyl-o-phospho-L-tyrosine
Eicosapentaenoic acid derivatives according to the present invention include the following compounds:
13. N-eicosapentaenoyl-o-phospho-2-aminoethanol
14. N-eicosapentaenoyl-o-phospho-L-serine
15. N-eicosapentaenoyl-o-phospho-L-threonine
16. N-eicosapentaenoyl-o-phospho-L-tyrosine
Linoleic acid derivatives according to the present invention include the following compounds:
17. N-linolenoyl-o-phospho-2-aminoethanol
18. N-linolenoyl-o-phospho-L-serine
19. N-linolenoyl-o-phospho-L-threonine
20. N-linolenoyl-o-phospho-L-tyrosine
The structural formulas of these compounds are presented below: 
In developing the scheme of synthesis the present inventor took into account the following properties of the title compounds:
i) a high degree of unsaturation
ii) presence of an asymmetric centre
iii) presence of sufficiently labile groups e.g. amide and phosphate.
Many of the well-known methods of acylation and phosphorylation could therefore not be used for the synthesis of such compounds. The preparation of the mixed carbonic carboxylic anhydrides followed by acylation of amino moiety of the methyl esters of hydroxyamino acids and ethanolamine was carried out at low temperature. The method of synthesis developed by the present inventor enables the N-acyl derivatives to be obtained with nearly quantitative yields. It is demonstrated that beta-cyanoethyl phosphate is a universal phosphorylation agent for hydroxyamino acids containing primary, secondary or aromatic OH-groups. Beta-cyanoethyl and ester protective groups were removed simultaneously by mild alkaline hydrolysis (0xc2x0 C., 1.5 N NaOH). It is worth emphasising that all compounds prepared by this method are formed in good or reasonable yields. In all experiments, the inventor has used synthesised title compounds in the form of ammonia salts.
The molecules of the title compounds have a polar and non-polar part (hydrophilic and hydrophobic in water systems) simultaneously. These long chain molecules are present in water solutions in the form of micelles, if their concentration is equal to the critical micelle concentration or higher. The structural organisation of the title compounds in micelles could be considered as a new organisation level of biological active molecules with specific physiological activity. It shall be noted that the process of micelle formation is reversible.
The influence of the title compounds 1 through 4, 1a through 4a, and 5 through 20 on humoral immune response was estimated by counting the quantity of antibody-forming cells (AFC) in the mice spleen. It has been experimentally proved, that the immunogenic activity of all compounds is dose-dependent. In particular, compounds 8, and 10 through 14, 17, 19 and 20, in doses of 45.5 and 91 xcexcg/mouse exhibit no immunogenic activity, but at the same time in the dose of 136.5 xcexcg/mouse these compounds displayed an immunostimulating action. It was found that the relative amount of AFC on the 5th day after injection of compound 10 increased by 138% and the total AFC amount increased by 159%. For compound 11xe2x80x94an increase of 67% and 80%, respectively, was recorded. For compound 12xe2x80x94an increase of 134% and 99% respectively, was recorded. For compound 8xe2x80x94an increase of 31% and 91% respectively, was recorded. For compound 13xe2x80x94an increase of 41% and 49% respectively, was recorded, and for compound 14xe2x80x94an increase of 52% and 51% respectively. For compound 17, the increase was 31% and 30%; for compound 19, 40% and 40%; for compound 20, 49% and 51%, respectively. The increase was calculated as compared with the amount of cells in the animals immunised only with sheep erythrocytes.
Compounds 3, 4, 1a, and 2a, in the doses of 45.5 xcexcg/mouse, exhibit no immunogenic activity. It was found that the relative amount of AFC on the 5th day after injection to the animals, for the compound 3, in a dose of 91 and 136.5 xcexcg/mouse, increased by 103% and 136%; and in respect of the total AFC amount an increase of 49%; and 164%, respectively, was recorded. For compound 4 in a dose of 91 and 136.5 xcexcg/mouse, the relative amount of AFC increased by 70% and 233% and the total AFC amount increased by 45% and 130%, respectively. For compound 1a in a dose of 91 and 136.5 xcexcg/mouse, the relative amount of AFC increased by 29% and 52%, and the total AFC amount increased by 27% and 66%, respectively. For compound 2a in a dose of 91 and 136.5 xcexcg/mouse, the relative amount of AFC increased by 93% and 111%, and the total AFC amount increased by 48% and 71%, respectively. The increase is calculated as a comparison with the amount of cells in animals immunised only with sheep erythrocytes.
The critical micelle concentration (CMC) values for compounds 1 through 4, 1a through 4a were determined as described by Griess W. (Fette Seifen Anstrichmi., 1955. Bd.57, s. 24-32) and for compounds 5 through 20 the CMC values were determined as described by A. Chattopadhyay and E. London (Anal. Biochem., 1984, 139, P.408-412). The CMC-values for ammonia salts of compounds 1 through 4 and 1a through 4a are in the interval of 1xc2x710xe2x88x924 M-4xc2x710xe2x88x924 M and for ammonia salts of compounds 5 through 20, in the interval of 1xc2x710xe2x88x923 M-2xc2x710xe2x88x923 M. That explains why, in the present experiments, compounds 1a, 2a, 3, and 4 display immunostimulating effect in vivo in micelle form at 91 xcexcg/mouse and compounds 8, and 10 through 14, 17, 19 and 20xe2x80x94at 136.5 xcexcg/mouse.
According to the obtained results, the title compounds display immunostimulating effects in vivo resulting in the enhancement of the amount of antibody-forming cells (AFC) in the spleen and in the enhancement of the antibody titres. Some of these compounds induce the conversion of B-lymphocytes to AFC and enhance the production of IgM-antibodies, ensuring a 2 to 3-fold increase of the immune response in C57B1/6 mice which are low-reactive to sheep red blood cells (SRBC).
It is known that one of the factors of genetically determined low reactivity of some lines of mice is insufficient extent of the processes of T- and B-lymphocyte migration and co-operation in immunisation with the particular antigen. Thus, using the studied compounds made it possible to convert genetically low-reacting animals to high-reacting ones. Lymphokines and monokines such as interleukin-1 and -2, thymic hormones, hematopoetin, factor of tumour necrosis, interferons, the molecular weight of which is less then that of albumin, are captured actively by the liver, filtrated by the kidneys, which results in their quick disappearance from the blood stream. The interest attracted by these immunomodulators is not too great due to their low effectiveness and high toxicity. The studied compounds can be promising as potent means for correction of immunity disorders localised at the level of immunocompetent cells, including the possibility of phenotypic correction of the immune response.
Reversible protein phosphorylation is one of the most important mechanisms of regulating intracellular processes such as cell cycle, growth and differentiation (Edelman A. M. et al., A. Rev. Biochem. V. 56., P. 567-613, 1987; Han K.-K., Martinage A., Int. J. Biochem. V. 24. N1.,P. 19-28, 1992). The most common phosphorylated amino acids are serine, threonine and tyrosine (on the hydroxyl group). Calcium and cAMP exert many of their cellular effects by controlling the activity of a protein kinases. Protein kinases catalyse the transfer of phosphate groups from a molecule of adenosine triphosphate (ATP) to other proteins. The addition of a phosphate group alters protein function; indeed, widespread protein phosphorylation is thought to underlie the changes in cell behaviour induced by some extracellular signals. It is contemplated by the present inventor that the title compounds could be such signal molecules.
Alpha-fetoprotein (AFP) forms a reversible equilibrium complex with N-arachidonoyl aminoethylphosphate (N-AAP). The protein is reversiblylinked with to a micelle containing up to 300 molecules of N-AAP (See International Application No PCT/EP99/04201 by the same inventor).
The inventor has now shown that AFP forms reversible equilibrium complexes with all the title compounds of the present invention. These inventive complexes may contain their components in highly varying molar ratios, such as from an equimolar ratio to a significant overabundance of compounds 1 through 4, 1a through 4a, and 5 through 20 in relation to AFP. For compounds 1 through 4, 1a through 4a, and 5 through 20, the inhibition equilibrium association constants (Ki) of arachidonic acid with AFP were determined to be in the intervalxe2x80x940.9xc2x7106 Mxe2x88x921-4xc2x7106 Mxe2x88x921.
The inventive complexes may be obtained by adding water solution of any titled compound to a water solution of AFP followed by ultrafiltration, said filtration resulting in concentrating the solution and removing any compound (e.g. any one of compounds 1 through 4, 1a through 4a, 5 through 20) that remained unbound to AFP. The AFP concentration in solution varies from 0, 1 to 2 mg/ml and that of the compound from 0,005 up to 30 mg/ml. The changes of molecular weight of the AFP as judged by gel-filtration is an evidence of the existence of AFP complexes with micelles of compounds 1 through 4, 1a through 4a, and 5 through 20.
In one embodiment the molecular weight of AFP incorporated in the complex with the title compound increases by approximately 2 times. Micelles contained about 100-200 lipid molecules. In another embodiment the molecular weight of AFP incorporated in the complex with any one of the compounds 1 through 4, 1a through 4a, and 5 through 20 increases by approximately 2-3 times. The micelles contained 200-300 lipid molecules.
The influence of complexes of AFP with one of the title compounds 1-4, 1a-4a, 5-20 as well as their basic components on humoral immune response was estimated by counting the quantity of antibody-forming cells (AFC) in the mice spleen. In particularly it has been experimentally proved that a compounds 1, 2a, 3 in the dose of 45.5 xcexcg/mouse and a compounds 7, 10, 14, 15 in the dose 91 xcexcg/mouse and human or rat AFP in the dose 9 xcexcg/mouse do not inhibit immunogenic activity.
Administration of a complex of AFP with compound 1 in the dose of 45.5 xcexcg/mouse (complex 1:100) resulted in that the relative amount of AFC increased by 56% and the total AFC amount increased by 43%; for complex AFP with compound 2a in the dose of 45.5 xcexcg/mouse (complex 1:100) the corresponding figures increased by 77% and 43% accordingly; for a complex APP with compound 3 in the dose of 45.5 xcexcg/mouse (complex 1:100) the corresponding figures increased by 76% and 48% accordingly in comparison with the amount of cells in animals immunized only with sheep erythrocytes.
Administration of a complex of AFP with compound 7 in the dose of 91 xcexcg/mouse (complex 1:200) resulted in that the relative amount of AFC increased by 49% and the total AFC amount increased by 47%; for complex AFP with compound 10 in the dose of 91 xcexcg/mouse (complex 1:200) the corresponding figures increased by 88% and 79% accordingly; for a complex AFP with compound 14 in the dose of 91 xcexcg/mouse (complex 1:200) the corresponding figures increased by 55% and 53% accordingly; for a complex AFP with compound 15 in the dose of 91 xcexcg/mouse (complex 1:200) the corresponding increased by 50% and 48%, respectively. The increase is calculated as a comparison with the amount of cells in animals immunised only with sheep erythrocytes.
All title compounds in the form of complexes with AFP exhibit the immunostimulating effect in vivo in lower concentrations than these compounds alone. It is suggested, taht AFP reduces the critical micelle concentration (CMC) of the title compounds on account of highly cooperative specific interactions in complexes comprising AFP and a ligand. So, in the experiments performed by the inventor, compounds 1, 2a, and 3 in a complex with AFP displayed an immunostimulating effect in vivo at a dose of 45.5 xcexcg/mouse and compounds 7, 10, 14, and 15xe2x80x94at a dose of 91 xcexcg/mouse.
In the experiments disclosed in the present application, the inventor has used C57B1/6 mice, which are low reactive to sheep red blood cells (SRBC). It possible, that complexes title compounds 1-4, 1a-4a, and 5-20 with AFP would display immunostimulating effect at more low amounts, if mice, which are high reactive to SRBC, have been used. The present inventor has shown that complex compound 5 (N-AAP) with AFP displays immunostimulating effect at 45.5 xcexcg/mouse, in experiments with CBA mice (See the International Application No PCT/EP99/04201).
AFP was isolated from human cord blood by immunoaffinity chromatography on monoclonal antibodies against AFP immobilised on Sepharose(copyright) immunoaffinity chromatography on polyclonal antibodies to the proteins of normal human blood and gel-filtration on Sephacryl S-200(copyright). The AFP preparation thus obtained exhibited a purity above 99% and did not contain low molecular weight impurities and retained completely its biological activity.
Rat AFP was isolated from neonatal rat serum. Monospecific anti-rat serum alfa-fetoprotein IgG was coupled to cyanogen bromide-activated Sepharose(copyright) 4B (4.5 mg/ml packed volume of gel) to yield an immunoaffinity matrix. Acidic elution conditions were as described previously (Calvo M., et al., J.Chromatogr. Vol.328, p. 392-395, 1985).
Other sources of AFP may be purified and/or modified AFP from other mammals, for example from genetically modified mammals, or from cell cultures. Preferably, the AFP is biotechnologically manufactured using a cell culture of genetically modified cells expressing human AFP. With knowledge of the nucleotide sequence coding for human AFP, this can be inserted in a host, together with necessary promoters and other sequence information, for example sequences influencing the extracelluar expression of AFP. The AFP is collected from the cell culture and purified by chromatography and may be further purified by gel-filtration. In any case the production method must involve steps, which guarantee that the final product is free from pyrogens and possible viral or bacterial contaminants. Suitable production methods can for example be found in the field of interferon production.
The novel retinoids according to the present invention exhibit good solubility in water due to the presence of the phosphate groups in the molecules. The water solubility opens many opportunities for obtaining various medical forms of the preparations possessing suitable properties for transdermal and/or oral absorption.
The compounds according to the invention can be used as such or as components in pharmaceutical compositions. The compounds can be given systemically or locally, for example topically. Suitable modes of administration of the compounds include intravenous administration, intraperitoneal administration, as well as oral, rectal and transdermal administration. The intended mode of administration is naturally taken into account in the preparation of the final pharmaceutical composition. Normal pharmaceutical adjuvants can naturally be used and the compounds can be made available in the form of injectable solutions, ointments, capsules or tablets, in the form of transdermal patches or suppositories according to the intended use and/or mode of administration.
The therapeutic effective doses for intravenous administration of the title compounds are in intervals:
Compounds 1, 1a, 5, 9, 13, 17: from 5 mg/kg to 10 mg/kg
Compounds 2, 2a, 3, 3a, 6, 7, 10, 11, 14, 15, 18, 19: from 5 mg/kg to 20 mg/kg
Compounds 4, 4a, 8, 12, 16, 20: from 5 mg/kg to 30 mg/kg
The therapeutic effective doses for intravenous administration complexes of AFP with title compounds are in intervals:
Compounds 1, 1a, 5, 9, 13, 17: from 2 mg/kg to 10 mg/kg
AFP: from 0.2 mg/kg to 1 mg/kg
Compounds 2, 2a, 3, 3a, 6, 7, 10, 11, 14, 15, 18, 19: from 2 mg/kg to 20 mg/kg
AFP: from 0.2 mg/kg to 2 mg/kg
Compounds 4, 4a, 8, 12, 16, 20: from 2 mg/kg to 30 mg/kg
AFP: from 0.2 mg/kg to 3 mg/kg